1. Field of the Invention
The present invention relates to treating cancer that overexpresses TopBP1 by administering to a patient suffering from the cancer with an effective amount of a small molecule inhibitor that binds the BRCT7/8 domain of TopBP1.
2. Description of the Related Art
Despite the complexity of mutations in different cancers, recent efforts in cancer genome sequencing project have shown a handful of core signaling pathways, such as receptor tyrosine kinases (RTK)/RAS/phosphatidylinositol 3-kinase (PI(3)K), p53 and retinoblastoma (Rb) protein, are deregulated in majority of solid tumors. For example, 77% of breast cancers have genetic alterations in PI(3)K/Akt pathway, and 49% have alterations in p53 signaling (see reference in (TCGA 2012)). Particularly in basal-like breast cancer (often triple-negative breast cancer or TNBC), 84% show TP53 mutations, 35% show PTEN mutation/loss and 20% show RB1 mutation/loss (see reference in (TCGA 2012)). These deregulated signaling pathways often converge to some common modulators.
As a key regulator for cell growth, the Rb pathway is de-regulated in most cancers, resulting in high E2F1 activities to drive cell cycle progression. E2F1 also has a pro-apoptotic role through activating target genes such as p73 (Irwin et al. 2000; Stiewe and Putzer 2000), Apaf-1, and caspases (Muller et al. 2001; Nahle et al. 2002) during DNA damage (Lin et al. 2001). How to activate E2F1 pro-apoptotic activity inside cancer cells remains an elusive goal. Previously, we showed that a checkpoint activator protein, TopBP1 (topoisomerase IIβ-binding protein 1), plays a critical role in suppressing E2F1 pro-apoptotic activity in response to PI(3)K/Akt signaling, which suggests TopBP1 as a therapeutic target to activate E2F1-dependent apoptosis in cancer (Liu et al. 2003; Liu et al. 2004; Liu et al. 2006; Liu et al. 2013).
TopBP1 utilizes its multiple BRCA1 carboxyl-terminal (BRCT) motifs as scaffolds to modulate many processes of DNA metabolism; such as DNA damage checkpoint, replication, and transcription (Garcia et al. 2005). TopBP1 represses E2F1 transcriptional activities by recruiting Brg1/Brm chromatin remodeling complex (Liu et al. 2004). TopBP1 also binds the DNA-binding domain (DBD) of p53 to inhibit its transcriptional function (Liu et al. 2009). Regulation of E2F1 and p53 by TopBP1 is important to control the pro-apoptotic activities of both transcription factors during normal S phase transition. While TopBP1 is involved in seemingly separate functions, our recent study showed that its functions in replication checkpoint and transcriptional regulation are indeed coordinated via an Akt-dependent conformational change of TopBP1 (Liu et al. 2013). Akt phosphorylates TopBP1 at Ser1159 and induces its oligomerization through an intermolecular interaction between the phosphorylated Ser1159 residue (pS1159) and the 7th-8th BRCT (BRCT7/8) domain of two TopBP1 molecules (Liu et al. 2006; Liu et al. 2013). Oligomerization of TopBP1 then induces its binding to E2F1, but at the same time prevents its recruitment to chromatin and ATR binding and perturbs its checkpoint-activating functions (Liu et al. 2013). Thus, by regulating TopBP1 quaternary structure, Akt switches TopBP1 function from checkpoint activation to transcriptional regulation. This mechanism is responsible for inhibition of E2F1-dependent apoptosis (an oncogenic checkpoint) and inhibition of ATR function (replication checkpoint) in the tumors with high Akt activity. Therefore, selective blockade of TopBP1 oligomerization may provide a novel therapeutic strategy in cancer cells which exhibit up-regulated PI3K/Akt signaling.
Many mutant p53 (mutp53) proteins do not only lose normal p53 function, but also gain new functions which contribute to cancer progression (“gain of function” activities (GOF)) (Freed-Pastor and Prives 2012; Muller and Vousden 2013). In addition to mutations, cancer cells can have a different mechanism to inactivate p53: up-regulation of p53 negative regulators, such as MDM2 (Manfredi 2010), MDMX (Marine et al. 2006) and TopBP1 (Liu et al. 2009). Adding to the complexity of p53 regulation is the presence of 12 p53 isoforms with differential expression (Khoury and Bourdon 2011), some of which have dominant-negative activities against p53 (Bourdon et al. 2005).
TopBP1 also mediates mutp53 GOF by facilitating its complex formation with NF-Y and p63/p73 (Liu et al. 2011). Since TopBP1 is an E2F target (Liu et al. 2004), it is often up-regulated upon inactivation of the Rb pathway (Liu et al. 2009). Indeed, TopBP1 is frequently overexpressed in breast cancer and its overexpression is associated with a shorter survival (Liu et al. 2009; Liu et al. 2011). SNPs in TopBP1 that cause higher expression of TopBP1 mRNA and protein have also been associated with an increased risk in breast and endometrial cancers (Forma et al. 2013a; Forma et al. 2013b). Thus, deregulation of the Rb pathway may be functionally linked to mutp53, and be responsible for at least part of mutp53 GOF via TopBP1. The accumulated TopBP1 in cancer cells then inhibits growth checkpoints through repressing E2F1 and p53 functions, and collaborates with mutp53 to further promote tumor progression. Therefore, TopBP1 might be a target among the nexus of these major oncogenic pathways. Here we perform a molecular docking screening and identify calcein as a compound to target the BRCT7/8 domain of TopBP1. We also use its cell-permeable derivative Calcein AM to provide proof-of-principle evidence for targeting TopBP1 as a cancer therapy.